Fig. 4

3D microniche significantly improved endothelial differentiation efficiency and helped to maintain long-term cell survival. a Protocol for endothelial differentiation from hESC in 3D microniches. b Bar graph quantification of CD31+ cell number obtained from 2D and 3D cultures on day 15. c Cell numbers at different times and culture medium used in 2D and 3D are listed in the table (n = 3, ^*** p < 0.001). d Immunostaining showing CD31 expression in 2D condition on differentiation day 10. Scale bars: 100 μm. e Immunostaining showing CD31 expression in 3D microniches on differentiation day 10 visualized by multiphoton microscopy. Scale bars: 100 μm. f Percentage of CD31⁺ cells during endothelial differentiation under 2D condition and 3D microniches on day 10 and day 27 (n = 3, ^*** p < 0.001). g ECs differentiated from hiPSCs were immunostained with VE-cadherin and CD31 antibodies. Scale bars: 100 μm. h and i ECs differentiated from hiPSCs in 3D GMs were digested out and tested in DiI-Ac-LDL uptake and tube formation assay. Scale bars: 100 μm